Technologies are needed that enable comprehensive biomarker and target discovery for detection, prognosis, patient stratification, and therapeutics. Methods of detecting biomolecules (e.g., polypeptides, nucleic acids, polysaccharides, and lipids) are widely used in a variety of fields including, e.g., proteomics and genomics. Detection, analysis, and identification of biomolecules can play an important role in the understanding of the biology of organisms and the causes of disease. (See, e.g., Pandey et al., Nature, 405: 837–846 (2000).
A number of different technologies have been developed to separate, analyze and identify biomolecules such as proteins. Typically, identification by mass spectrometry (MS) involves analysis of isolated proteins or peptide fragments, followed by mapping or tandem MS to obtain sequence information. One strategy that has been used to differentiate the resulting spectra involves tagging the proteins with reagents having different masses (“mass tags”). The most predominant mass tags are based on the mass difference of the isotopes hydrogen and deuterium. The isotopically distinct mass tags are referred to as Isotope-Coded Affinity Tags (ICAT), and their use allows a number of different samples to be analyzed at the same time and directly compared. See, e.g., Ranish et al. Nature Genet. 33: 349–355 (2003); Zhou et al., Nature Biotechnol. 19: 512–515 (2002); Gygi et al., J. Proteome Res. 1: 47–54 (2002); Gygi et al., Nature Biotechnol. 17: 994–998 (1999); Gygi and Aebersold, Curr. Opin. Chem. Biol. 4: 489–494 (2000); Aebersold and Mann, Nature 422: 198–207 (2003); Patterson and Aebersold, Nature Genetics Suppl. 33: 311–323 (2003); and Tao and Aebersold, Curr. Opin. Biotechnol. 14: 110–118 (2003); and WO 00/11208. The reagent consists of biotin for affinity selection, a linker that contains light (hydrogen) or heavy (deuterium) isotopes of hydrogen for mass tagging, and a Cys-reactive group (iodoacetamide) to derivatize proteins. Differential labeling involves using two isotopic reagents for two samples in comparative profiling. Samples are mixed following the ICAT derivatization step and proteolyzed together. The tagged peptides are affinity purified using an avidin column, and analyzed by mass spectrometry. The ratio of mass peak amplitude of peptides from proteins differentially labeled with heavy and light mass tags gives a measure of the relative amounts of each protein. The ICAT method, using a heavy reagent and a light reagent, is limited to differential analysis of two samples.
ICAT has a number of shortcomings. First, ICAT utilizes only two different masses (light and heavy). Thus, the method is limited to applications that require comparisons of only two states. Second, cysteine (Cys) is an amino acid of low abundance (about 2.2%). Moreover, many cells contain endogenously biotinylated proteins, the proteolyzed fragments of which are immobilized by the affinity column. Of particular note is the tendency of the deuterated and non-deuterated probes to elute differentially, giving rise to more than one peak. Finally, the biotinylated tags have a tendency to fragment during mass spectrometric analysis.
In view of the shortcomings of tagging methodologies as presently practiced, there is a need in the art for methods of detecting, analyzing, and identifying biomolecules in a sample, including those present only in small quantities. A method that was more versatile and robust than those methods based upon ICAT would overcome current limitations in biomolecule analysis. The present invention provides such a method.